Ammonia synthesis methods and systems

ABSTRACT

Systems and methods for producing ammonia are described. In one embodiment, hydrogen, carbon dioxide, and nitrogen are dissolved in a solution. A glutamine synthetase inhibitor and autotrophic diazotroph bacteria are also placed in the solution.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under GrantN00014-11-1-0725 awarded by the Office of Naval ResearchMultidisciplinary University Research Initiative, and GrantFA9550-09-1-0689 awarded by The Air Force Office of Scientific Research.The government has certain rights in the invention.

FIELD

Disclosed embodiments are related to ammonia synthesis.

BACKGROUND

Due to its use and large-scale agriculture, the reduction of N₂ into NH₃is essential in maintaining the global geochemical nitrogen cycle andthe sustainability of the human population. The most common method forproducing industrial scale quantities of NH₃ is the industrialHaber-Bosch process. The Haber-Bosch process is efficient and scalable.However, this process consumes large volumes of natural gas asfeedstock, operates at high temperature and pressure, and relies on acentralized production and subsequently transport for NH₃ distribution.

SUMMARY

In one embodiment, a method for producing ammonia includes: dissolvinghydrogen in a solution; dissolving carbon dioxide in the solution;dissolving nitrogen in the solution; placing a glutamine synthetaseinhibitor in the solution; and placing autotrophic diazotroph bacteriain the solution.

In another embodiment, a system for producing ammonia includes a reactorchamber with a solution contained therein. The solution includesdissolved hydrogen, dissolved carbon dioxide, dissolved nitrogen, aglutamine synthetase inhibitor, and autotrophic diazotroph bacteria.

It should be appreciated that the foregoing concepts, and additionalconcepts discussed below, may be arranged in any suitable combination,as the present disclosure is not limited in this respect. Further, otheradvantages and novel features of the present disclosure will becomeapparent from the following detailed description of various non-limitingembodiments when considered in conjunction with the accompanyingfigures.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures may be represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 is a schematic of distributed ammonia synthesis at ambientconditions within a reactor;

FIG. 2 is a graph of N₂ reduction using the CoPi|Co—P| X. autotrophicuscatalyst system with OD₆₀₀, the amount of charge passed through, theconcentration of total nitrogen content (N_(total)), and solublenitrogen content (N_(soluble)) plotted vs. time;

FIG. 3 is a graph of change of N_(total) and OD₆₀₀ under differentconditions;

FIG. 4 is a graph of linear scan voltammetry (line, 10 mV/sec) andchronoamperometry (circle, 30 min average) of Co—P HER cathode in X.autotrophicus medium, iR corrected;

FIG. 5 Is a schematic diagram of NH₃ production in an extracellularmedia; and

FIG. 6 is a graph of OD₆₀₀, the amount of charge passed through, theconcentration of total nitrogen content (N_(total)) and NH₃/NH₄ ⁺extracellular content (NH₃) plotted against time.

DETAILED DESCRIPTION

Unlike more traditional production methods, catalytic NH₃ synthesis fromN₂ has been reported with transition metal complexes, electrocatalysts,photocatalysts, nitrogenase, and heterotrophic diazotrophs. However,these approaches typically provide limited turnovers and use sacrificialchemicals as reductants. Consequently, the Inventors have recognizedthat it may be desirable to enable a selective NH₃ synthesis from N₂ andH₂O at ambient conditions. This may help enable a distributed approachtowards NH₃ synthesis at ambient conditions, which may also beintegrated with different forms of power including renewable energysources. Possible benefits associated with such a production approachmay include enabling on-site production and deployment of ammonia whilealso reducing CO₂ emissions as compared to more traditional productionmethods.

In view of the above, the Inventors have recognized the benefitsassociated with using a reactor-based arrangement including a solutionwith one or more types of bacteria that include one or more enzymesuseful in the production of ammonia. Specifically, in one embodiment, asystem for producing ammonia may include a reactor with a chambercontaining a solution. The solution may include dissolved hydrogen,carbon dioxide, and nitrogen as well as a glutamine synthetase inhibitorin the solution. The solution may also include one or more forms ofautotrophic diazotroph bacteria in the solution. During use, theautotrophic diazotroph bacteria metabolize compounds within the solutionto produce ammonia. Specifically, the bacteria may include nitrogenase,such as RuBisCO, and hydrogenase enzymes that utilize nitrogen, carbondioxide, and hydrogenase to form the desired ammonia. Appropriateautotrophic diazotroph bacteria include Xanthobacter autotrophicus,Bradyrhizobium japonicum, or any other appropriate bacteria capable ofmetabolizing the noted compounds to produce ammonia.

Depending on the embodiment, an inhibitor may be included in a solutionto at least partially prevent the uptake of ammonia into the biomass ofthe bacteria. Thus, at least a portion of the ammonia produced by thebacteria may be excreted into the solution for subsequent collection. Inone specific embodiment a glutamine synthetase (GS) inhibator such asglufosinate (PPT), methionine sulfoximine (MSO), or any otherappropriate inhibitor may be used.

In some embodiments, a solution placed in the chamber of a reactor mayinclude water with one or more additional solvents, compounds, and/oradditives. For example, the solution may include: inorganic salts suchas phosphates including sodium phosphates and potassium phosphates;trace metal supplements such as iron, nickel, manganese, zinc, copper,and molybdenum; or any other appropriate component in addition to thedissolved gasses noted above. In one such embodiment, a phosphate mayhave a concentration between 9 and 50 mM.

The above noted concentrations of dissolved gases may be controlled inany number of ways including bubbling gases through the solution,generating the dissolved gases within the solution (e.g. electrolysis),or any other appropriate method of controlling the concentration ofdissolved gas within the solution. Additionally, the various methods ofcontrolling concentration may either be operated in a steady-state modewith constant operating parameters, and/or a concentration of one ormore of the dissolved gases may be monitored to enable a feedbackprocess to actively change the concentrations, generation rates, orother appropriate parameter to change the concentration of dissolvedgases to be within the desired ranges noted above. Monitoring of the gasconcentrations may be done in any appropriate manner including pHmonitoring, dissolved oxygen meters, gas chromatography, or any otherappropriate method.

In some embodiments, hydrogen may be provided to a solution using theelectrolysis of water, i.e. water splitting. Depending on the particularembodiment, a power source may be connected to a first electrode and asecond electrode that are at least partially immersed in a solutionwithin a reactor chamber. The power source may correspond to anyappropriate source of electrical current that is applied to theelectrodes. However, in at least one embodiment, the power source maycorrespond to a renewable source of energy such as a solar cell, windturbine, or any other appropriate source of current though embodimentsin which a non-renewable energy source is used are also contemplated. Ineither case, a current from the power source is passed through theelectrodes and solution to evolve hydrogen and oxygen. The current maybe controlled to produce a desired amount of hydrogen and/or oxygenproduction at a desired rate of production. In one embodiment, theelectrodes may be coated with, or formed from, a water splittingcatalyst to further facilitate water splitting and/or reduce the voltageapplied to the solution. For example, the electrodes may be made fromone or more of a cobalt-phosphorus alloy, cobalt phosphate, cobaltoxide, cobalt hydroxide, cobalt oxyhydroxide, or any other appropriatematerial. In one specific embodiment, the first and second electrodesmay correspond to a cathode including a cobalt-phosphorus alloy and ananode including cobalt phosphate. However, embodiments in which othertypes of anodes and/or cathodes are used are also contemplated as thedisclosure is not so limited.

In instances where a phosphorus based anode and/or cathode is used, suchas a cobalt-phosphorus alloy and/or a cobalt phosphate, a phosphatebuffer may be included in the solution. Appropriate phosphates include,but are not limited to, sodium phosphates and potassium phosphates.Without wishing to be bound by theory, it is believed that duringelectrolysis of the water, phosphorus and/or cobalt is extracted fromthe electrodes. The reduction potential of leached cobalt is such thatformation of cobalt phosphate from phosphate available in the solutionis energetically favored. Cobalt phosphate formed in solution thendeposits onto the anode at a rate linearly proportional to free cobaltphosphate, providing a self-healing process for the electrodes. Aconcentration of phosphate may be between 9 and 50 mM though otherconcentrations may also be used as the disclosure is not so limited.

In embodiments where hydrogen is produced using water electrolysis, avoltage applied to a pair of electrodes immersed in a solution may belimited to be between first and second voltage thresholds. In one suchembodiment, the voltage applied to the electrodes may be greater than orequal to about 1.8 V, 2 V, 2.2 V, 2.4 V, or any other appropriatevoltage. Additionally, the applied voltage may be less than or equal toabout 3 V, 2.8 V, 2.6 V, 2.4 V, or any other appropriate voltage.Combinations of the above noted voltage ranges are contemplatedincluding, for example, a voltage applied to a pair of electrodes thatis between 1.8 V and 3 V. However, it should be understood that voltagesboth greater than and less than those noted above, as well as differentcombinations of the above ranges, are also contemplated as thedisclosure is not so limited. For example, it is envisioned that othercatalysts that enable a water splitting voltage closer to the idealsplitting voltage of 1.23 V may also be used.

As noted previously, in some embodiments, a flow of gas may beintroduced to a solution contained within a reactor chamber to dissolvea desired ratio of gases in the solution. For example, in oneembodiment, a system may include one or more gas sources that arefluidly connected to one or more gas inlets associated with the chamber.The gas inlets are arranged to bubble the gas through the solution. Forexample, a one-way valve may be fluidly connected to an inlet to thechamber bottom, a tube connected to a gas source may have an endimmersed in the solution within the chamber, or the system may use anyother appropriate arrangement to introduce the gases to the solution.Thus, when a gas source provides a pressurized flow of gas to thechamber, the gas is introduced into the solution where it bubbles upthrough the solution dissolving at least a portion of the gas therein.

While a gas source may correspond to any appropriate type of gas, in oneembodiment, a gas source may provide one or more of hydrogen, nitrogen,carbon dioxide, and oxygen. Additionally, a total flow of gases providedby one or more gas sources to a solution within a reactor chamber mayhave any appropriate composition of gases. However, in one embodiment, aflow of gas may contain between 10 and 99.46% nitrogen, 0.04 and 90%carbon dioxide, and/or 0.5% and 5% oxygen. Of course embodiments inwhich a different mix of gases is bubbled through a solution includingdifferent gases and/or different concentrations both greater than andless than those noted above are also contemplated as the disclosure isnot so limited.

EXAMPLES

A reactor used in the experiments included a biocompatible watersplitting catalyst system including a cobalt-phosphorous (Co—P) alloycathode for the hydrogen evolution reaction (HER) and a cobalt phosphate(CoP_(i)) anode for the oxygen evolution reaction (OER). This systemenabled the use of a low driving voltage (E_(appl)) while producing thedesired hydrogen for use in producing ammonia. Specifically, NH₃synthesis from N₂ and H₂O was accomplished using the water splittingsystem and driving the N₂ reduction reaction within H₂-oxidizing,autotrophic microorganisms. In this case, Xanthobacter autotrophicus (X.autotrophicus) was used. X. autotrophicus is a gram-negative bacteriumthat belongs to a small group of diazotrophs, which at micro-aerobiccondition (less than about 5% O₂) can use H₂ as their sole energy sourceto fix CO₂ and N₂ into biomass. Therefore, in this experimental setup,electrochemical water splitting generated H₂ as the biological energysource and in the same reactor X. autotrophicus acted as theroom-temperature N₂ reduction reaction catalyst to convert H₂ and N₂into NH₃.

FIG. 1 shows a schematic of the experimental setup including asingle-chamber reactor that houses electrodes immersed in a watersolution. The electrodes included a Co—P cathode for the hydrogenevolution reaction and a CoP_(i) anode for the oxygen evolutionreaction. A gas mixture including 2% O₂, 20% CO₂, and 78% N₂ was bubbledthrough the solution at a flow rate of greater than or equal to 5 mL/minto maintain a micro-aerobic environment.

During the experiments, a constant voltage (E_(appl)) was appliedbetween the OER and HER electrodes for water splitting. The hydrogenases(H₂ases) of X. autotrophicus oxidized the generated H₂, fueling CO₂reduction in the Calvin cycle and N₂ fixation by nitrogenases (N₂ases).Each turnover of N₂ reduction yields two NH₃ and one H₂ molecule(s), thelatter of which may be recycled by the hydrogenases. The generated NH₃is typically incorporated into biomass, but can also diffuseextracellularly as a result of accumulation from inhibiting NH₃anabolism (pathway 2) as described previously.

At the beginning of each experiment, X. autotrophicus was inoculatedinto the organic-free minimal medium without any nitrogen supplement. Aconstant driving voltage (E_(appl)=3.0 V) was applied to theCoP_(i)|Co—P catalyst system, and aliquots were periodically sampled forthe quantification of biomass (optical density at 600 nm, OD₆₀₀) as wellas fixed nitrogen (colorimetric assay).

The CoP_(i)|Co—P| X. autotrophicus hybrid system used electricity toreduce N₂, as well as CO₂, into biomass without sacrificial reagents.FIG. 2 presents a graph of OD₆₀₀, the amount of charge passed through,the concentration of total nitrogen content (N_(total)), and solublenitrogen content (N_(soluble)) plotted versus the duration of theexperiments. The OD₆₀₀ in a H₂-fermentation experiment (“H₂ jar”) wasalso plotted as a comparison. The error bars in the graph denotestandard error of the mean (SEM) with n≥3. As shown in the figure, theamount of charge passed into water splitting was proportional to biomassaccumulation (OD₆₀₀) as well as the total nitrogen content in the medium(N_(total)) during the 5 day experiments.

FIG. 3 presents the change of N_(total) and OD₆₀₀ under differentexperimental conditions during the 5 day experiments. As seen in thefigure, the fixed nitrogen was assimilated into biomass, as there was nochange in the extracellular soluble nitrogen content (N_(soluble)). 72±5mg/L of N_(total), as well as 553±51 mg/L of dry cell weight,accumulated continuously over the experiment (n=3, entry 1 in FIG. 3).In contrast, no accumulation of N_(total) was observed in controls thatomitted one of the following elements in the design: water splitting, X.autotrophicus, a single-chamber reactor, and a microaerobic environment(entry 2 to 5 in FIG. 2b ). Particularly in the case of the dual-chamberexperiment (entry 4 in FIG. 3), the absence of N_(total) accumulation isconcurrent with the increase of soluble Co²⁺ concentration in the mediumfrom 0.9±0.2 μM to 40±6 μM within 24 hours as determined by inductivelycoupled plasma mass spectroscopy (ICP-MS), which is close to the ˜50 μMhalf maximum inhibitory concentration (IC₅₀) of X. autotrophicus.Without wishing to be bound by theory, this may indicate that theinstallation of an anion exchange membrane (AEM) prevented thedeposition of leached Co⁺ onto the CoP_(i) anode, illustrating that thebiocompatibility of the materials used in the system may be a desirablesystem property. As also illustrated in the figure, increases in OD₆₀₀that greatly exceed increases in N_(total) (entry 4 and 5 in 3) arelikely due to light scattering from the accumulation ofpoly(3-hydroxybutyrate), which is produced as a carbon storage polymerin conditions of nutrient constraints coupled with carbon excess.

The NRR activity of the described hybrid system is also supported bywhole-cell acetylene reduction assays that were done. Specifically,aliquots were sampled directly from operating devices that were exposedto an O₂/H₂/CO₂/Ar gas environment (2/10/10/78) and were able to reduceinjected C₂H₂ exclusively into C₂H₄ at a rate of 127±33 μM·h⁻¹·OD₆₀₀ ⁻¹(n=3). If the kinetic rate of C₂H₂ reduction by nitrogenase is onefourth of N₂ reduction based on the reaction stoichiometry, thisactivity corresponds to ˜12 mg/L N_(total) per day for cultures ofOD₆₀₀=1.0. This N₂-fixing rate is consistent with the measured N_(total)accumulation during the 5 day experiments and excludes the possibilitiesof other hypothetical nitrogen sources in conjunction with othercontrols (vide supra). This measurement corresponds to a NRR turnoverfrequency (TOF) of 1.4×10⁴ s⁻¹ per bacterial cell. If assuming anitrogenase copy number of about 5000 based on previous literature, thisNRR TOF corresponds to roughly ˜3 s⁻¹ per enzyme, which is consistentwith previous studies. The equivalent turnover number (TON) is roughly8×10⁹ per bacterial cell and 1×10⁶ per nitrogenase, at least 2 orders ofmagnitude higher than previously reported synthetic and biologicalcatalysts.

FIG. 4 presents the results from linear scan voltammetry (line, 10mV/sec) and chronoamperometry (circle, 30 min average) of Co—P HERcathode in X. autotrophicus medium, iR corrected. The thermodynamicvalues of HER and NRR (E_(HER), E_(NRR)) are displayed. Voltagecontributions from the applied E_(appl)=3.0 V is shown below the I-Vcharacterization. The NRR reaction operates with kinetic driving forcesas low as 160 mV. The I-V characteristics of the Co—P HER cathode in X.autotrophicus medium indicate an apparent overpotential of about 0.43 V.Without wishing to be bound by theory, much of this value is notintrinsic to the catalytic properties of the electrodes, but originatesfrom the build-up of a proton concentration gradient in the weaklybuffered solution (9.4 mM phosphate). By subtracting the contribution ofmass transport, the intrinsic NRR overpotential is about 0.16 V, muchlower than previous reports in literature. The dilute medium salinitysubsequently uses a driving voltage of E_(appl)=3.0 V, which is higherthan previous reported. The low ionic conductivity contributes to about28% of E_(appl)(˜0.85 V), which may likely be reduced by additionaloptimization. Regardless, the energy efficiency of NRR (η_(elec,NRR)) inthe experiments is 1.8±0.3% (n=3) during the 5 day experiments, inaddition to the 11.6±1.9% electrical CO₂ reduction efficiency(η_(elec,CO2), n=3). This corresponds to ˜900 GJ per tonne NH₃, whilethe thermodynamic limit is 20.9 GJ per tonne NH₃. Based on the reactionstoichiometry of nitrogenase and upstream biochemical pathways, thetheoretical number of H₂ molecules needed to reduce one N₂ moleculeranges in between 9.4˜14.7, which sets an upper bound of η_(elec,NRR) at7.5˜11.7%. Therefore, the amount of nitrogen reduction reported in thisexperiment is 15˜23% of the theoretical yield, much higher than thefaradaic efficiencies or quantum yields of other systems at ambientconditions.

The described experiments and systems exhibited faster N₂ reduction andmicrobial growth as compared to gas fermentation at similar conditions.In contrast to the observed linear growth in the hybrid system (FIG. 2),gas fermentation in the same conditions supplemented with a headspacecontaining ˜10% H₂ (“H₂ jar” experiment in FIG. 2) shows relativelyslow, nonlinear growth. This difference is dependent on N₂ fixation, asgrowth under gas fermentation and electrolysis demonstrated nodiscernable difference when ammonia is supplemented into the medium.Without wishing to be bound by theory, it is believed that this is theresult of competitive inhibition of H₂ on nitrogenase, with aninhibition constant K_(is)(D₂) of ˜11 kPa. Where electrolysis maintainsa low H₂ partial pressure at steady state in the hybrid device, the highH₂ concentration in gas fermentation may slow down the N₂ fixation rateand/or reduce the NRR energy efficiency. This hypothesis is supported bynumerical simulations, which show slower biomass accumulation in thecase of gas fermentation. Therefore, the current experiments indicatethat the described hybrid device can provide additional benefits ascompared to the simple combination of gas fermenters with awater-splitting electrolyzer, as the generated H₂ from water splittingcan influence downstream biochemical pathways.

The hybrid device is capable of excreting synthesized NH₃ into anextracellular medium. Previous biochemical assays and genome sequencingon this strain indicate that the NH₃ generated from nitrogenase isincorporated into biomass via a two-step process mediated by glutaminesynthetase (GS) and glutamate synthase (GOGAT) (FIGS. 1 and 5). If thefunctionality of this NH₃ assimilation pathway is disrupted, directproduction of an extracellular NH₃ fertilizer solution is realized. Ithas been reported that GS inhibitors can be used for NH₃ secretion insugar-fementating diazotrophs. As a proof of principle, glufosinate(PPT), a specific GS inhibitor commercially used as herbicide, was usedto block the NH₃ assimilation pathway and allow the synthesized NH₃ topassively diffuse out into the extracellular medium (pathway 2 in FIG.1, and FIG. 5). After the addition of PPT, the biomass of X.autotrophicus stagnated, while N_(t)w and the concentration of free NH₃in the solution (N_(NH3)) increased (FIG. 6). This indicates thatnitrogen accumulation after PPT addition mostly took the form ofextracellular NH₃. In the end of experiments, the concentration ofN_(NH3) was 11±2 mg/L (˜0.8 mM) and the accumulated N_(total) reached47±3 mg/L (n=3, Table S1). The rate of N₂ fixation tends to slow down inthe latter phase of the experiments, which may be related to nitrogenregulation at transcriptional and post-transcriptional levels. Furtherengineering in synthetic biology is capable of alleviating thislimitation.

The above experiments demonstrate the production and use of analternative NH₃ synthesis approach from N₂, H₂O, and electricity. Thewater splitting-biosynthetic process operates at ambient conditions andcan be distributed for an on-demand supply of nitrogen fertilizer. Whencoupled with a renewable energy supply such as a photovoltaic device of18% energy efficiency, solar-powered N₂ fixation into NH₃ can beachieved at up to a 0.3% solar-to-NH₃ efficiency along with a 2.1% solarCO₂ reduction efficiency. A typical cropping system annually reduces ˜11g nitrogen per m², which corresponds to a ˜4×10⁵ solar-to-NH₃ efficiency(assuming 2000 kWh/m² annual solar irradiance). Therefore, this approachyields a much higher efficiency and provides a sustainable route forfertilizer production without the use of fossil fuels. Though instancesin which the various feeds stocks (i.e. gases) could be provided usingfossil fuels as the current disclosure is not limited to only usingrenewable energies and/or splitting water directly in a reactor toproduce the desire ammonia generation.

While the present teachings have been described in conjunction withvarious embodiments and examples, it is not intended that the presentteachings be limited to such embodiments or examples. On the contrary,the present teachings encompass various alternatives, modifications, andequivalents, as will be appreciated by those of skill in the art.Accordingly, the foregoing description and drawings are by way ofexample only.

What is claimed is:
 1. A method for producing ammonia, the methodcomprising: dissolving hydrogen in a solution; dissolving carbon dioxidein the solution; dissolving nitrogen in the solution; placing aglutamine synthetase inhibitor in the solution; and placing autotrophicdiazotroph bacteria in the solution.
 2. The method of claim 1, whereindissolving hydrogen in the solution further comprises splitting water inthe solution to form hydrogen and oxygen.
 3. The method of claim 1,wherein splitting water in the solution further comprises splittingwater using a cathode including cobalt-phosphorus alloy and an anodeincluding cobalt phosphate.
 4. The method of claim 3, wherein thesolution includes a phosphate.
 5. The method of claim 1, whereindissolving carbon dioxide and nitrogen in the solution further comprisesbubbling carbon dioxide and nitrogen through the solution.
 6. A systemfor producing ammonia comprising: a reactor chamber with a solutioncontained therein, wherein the solution includes dissolved hydrogen,dissolved carbon dioxide, dissolved nitrogen, a glutamine synthetaseinhibitor, and autotrophic diazotroph bacteria
 7. The system of claim 6,further comprising a power source connected to a first electrodecomprising a cobalt phosphorus alloy and a second electrode comprisingcobalt phosphate, wherein the first electrode and the second electrodeare at least partially immersed in the solution within the reactorchamber.
 8. The system of claim 7, further comprising a phosphate in thesolution.
 9. Further comprising the system of claim 6, furthercomprising a gas inlet that bubbles gas through the solution within thereactor chamber.
 10. The system of claim 9, a gas source comprising atleast one of nitrogen, hydrogen, and carbon dioxide fluidly connected tothe gas inlet.